Effect of Huangjingwan on Expressions of Wnt/<italic style="font-style: italic">β</italic>-catenin Signal Pathway-associated Proteins in Hippocampus of Mice with Alzheimer's Disease Induced by <italic style="font-style: italic">D</italic>-galactose and Okadaic Acid with Learning and Memory Disorders

Hong-yue QIAN; Yi-sheng XIAO; Ji-hua HOU; Jie-lin JIANG; Qi ZHANG; Ai-ren ZUO; Meng GAO

doi:10.13422/j.cnki.syfjx.20201876

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Effect of Huangjingwan on Expressions of Wnt/β-catenin Signal Pathway-associated Proteins in Hippocampus of Mice with Alzheimer's Disease Induced by D-galactose and Okadaic Acid with Learning and Memory Disorders

更新时间：2021-12-03

- Effect of Huangjingwan on Expressions of Wnt/β-catenin Signal Pathway-associated Proteins in Hippocampus of Mice with Alzheimer's Disease Induced by D-galactose and Okadaic Acid with Learning and Memory Disorders
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 27, Issue 1, Pages: 63-71(2021)
- 作者机构：
  
  江西中医药大学中医学院，南昌 330004
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20201876
  CLC： R2-0;R22;R285.5;R289
- Received：05 April 2020，
  
  Published Online：27 July 2020，
  
  Published：05 January 2021
- 稿件说明：
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钱红月,肖移生,侯吉华等.黄精丸对D-半乳糖和冈田酸所致学习记忆障碍小鼠海马Wnt/β-catenin信号通路相关蛋白表达的影响[J].中国实验方剂学杂志,2021,27(01):63-71.

QIAN Hong-yue,XIAO Yi-sheng,HOU Ji-hua,et al.Effect of Huangjingwan on Expressions of Wnt/β-catenin Signal Pathway-associated Proteins in Hippocampus of Mice with Alzheimer's Disease Induced by D-galactose and Okadaic Acid with Learning and Memory Disorders[J].Chinese Journal of Experimental Traditional Medical Formulae,2021,27(01):63-71.
钱红月,肖移生,侯吉华等.黄精丸对D-半乳糖和冈田酸所致学习记忆障碍小鼠海马Wnt/β-catenin信号通路相关蛋白表达的影响[J].中国实验方剂学杂志,2021,27(01):63-71. DOI： 10.13422/j.cnki.syfjx.20201876.

QIAN Hong-yue,XIAO Yi-sheng,HOU Ji-hua,et al.Effect of Huangjingwan on Expressions of Wnt/β-catenin Signal Pathway-associated Proteins in Hippocampus of Mice with Alzheimer's Disease Induced by D-galactose and Okadaic Acid with Learning and Memory Disorders[J].Chinese Journal of Experimental Traditional Medical Formulae,2021,27(01):63-71. DOI： 10.13422/j.cnki.syfjx.20201876.

摘要

目的

探讨黄精丸对

-半乳糖和冈田酸所致学习记忆障碍造模的阿尔茨海默病（AD）小鼠海马Wnt

-连环蛋白（

-catenin）信号通路相关蛋白表达的影响，探讨其相关的作用机制。

方法

采用小鼠颈背部皮下注射1

-半乳糖液（0.14 g

-1

，连续4周）后，再右侧脑室一次性注射冈田酸2 μL（75 ng），制作小鼠AD模型，经Morris水迷宫检测挑选造模成功的AD小鼠，再随机分为AD模型组，美金刚组（1.3×10

-3

-1

），黄精丸组（2.5 g

-1

），另设假手术组和正常组；同时点给假手术组的小鼠右侧脑室一次性注射生理盐水2 μL处置作造模对照。造模2周后给两实验药物组小鼠灌胃相应剂量的试验药物，持续4周；另外，AD造模2周后，同时给假手术组及AD模型组小鼠每天灌胃等量生理盐水，持续4周；正常组小鼠无特殊处理。灌胃结束后，采用穿梭实验检测各组小鼠学习、记忆水平的区别；免疫组化检测各组小鼠海马CA1区

-catenin及糖原合成酶激酶3

（GSK-3

）阳性神经元数量改变；实时荧光定量聚合酶链式反应（Real-time PCR）检测各组小鼠海马GSK-3

，

-catenin，细胞周期蛋白D

（CyclinD

） mRNA表达差异；蛋白免疫印迹法（Western blot）检测各组小鼠海马总GSK-3

（t-GSK-3

），Ser9位磷酸化GSK-3

（p-Ser9-GSK-3

），Tyr216位磷酸化GSK-3

（p-Tyr216-GSK-3

），总

-catenin（t-

-catenin），磷酸化

-catenin（p-

-catenin）和CyclinD

蛋白表达变化。

结果

与正常组比较，AD模型组小鼠痴呆症状较明显，学习与记忆成绩明显降低，海马CA1区

-catenin免疫阳性神经元数量、海马t-

-catenin与CyclinD

mRNA及蛋白表达水平，p-Ser9-GSK-3

蛋白水平，p-Ser9-GSK-3

/t-GSK-3

及p-Tyr216-GSK-3

/t-GSK-3

值显著降低（

0.01），海马CA1区GSK-3

免疫阳性神经元数量、海马t-GSK-3

mRNA及蛋白表达水平，p-Tyr216-GSK-3

及p-

-catenin蛋白水平，p-

-catenin/t-

-catenin值均显著升高（

0.01）。与AD模型组比较，黄精丸组小鼠痴呆症状显著改善，学习与记忆成绩明显提高，海马CA1区

-catenin免疫阳性神经元数量、海马t-

-catenin，CyclinD

mRNA及蛋白表达水平，p-Ser9-GSK-3

蛋白水平，p-Ser9-GSK-3

/t-GSK-3

值均显著升高（

0.01），海马CA1区GSK-3

免疫阳性神经元数量、海马t-GSK-3

的mRNA及蛋白表达水平，p-Tyr216-GSK-3

及p-

-catenin蛋白水平，p-

-catenin/t-

-catenin值均显著下降（

0.01），但p-Tyr216-GSK-3

/t-GSK-3

值无明显统计学差异。

结论

黄精丸可下调AD小鼠海马t-GSK-3

表达并降低其蛋白活性，上调t-

-catenin表达并增加其蛋白活性，进而使下游CyclinD

表达增强，促进目的基因转录，发挥治疗AD作用，其机制可能与激活Wnt

-catenin信号通路有关。

Abstract

Objective

To explore the effect of Huangjingwan （HW） on the expressions of Wnt

-catenin signal pathway-associated proteins in the hippocampus of mice with Alzheimer's disease （AD） induced by

-galactose and okadaic acid with learning and memory disorders， as well as its mechanism.

Method

After subcutaneous injection with 1.0%

-galactose （0.14 g

-1

） into the back and neck of mice for 4 weeks， the right ventricle of mice was injected with 2 μL（75 ng） of okadaic acid for one time to make AD model， and the successfully modeled AD mice were selected by Morris water maze. Then， the selected AD mice were randomly divided into AD model group， memantine group （1.3×10

-3

-1

） and HW group （2.5 g

-1

）. In addition， the sham model control group and the normal control group were set up. At the same time， 2 μL normal saline was injected into the right ventricle of mouse in the sham model control group as the modeling control. Two weeks after molding， the mice in the two experimental drug groups were given the corresponding dose of the experimental drug by gavage for 4 weeks. In addition， after 2 weeks of AD modeling， mice in sham model control group and AD model group were intragastrically administrated with the same amount of normal saline daily for 4 weeks. There was no special treatment in the normal control group. At the end of gavage， the shuttle experiment was performed to detect the differences in learning and memory levels of mice in each group. The changes of

-catenin and GSK-3

positive neurons in CA1 area of hippocampus in each group were tested by immunohistochemistry. Quantitative real-time polymerase chain reaction （Real-time PCR） was used to measure the mRNA expressions of GSK-3

，

-catenin and CyclinD

in hippocampus of mice in each group. The Western blot was used to detect the expressions of total GSK-3

（t-GSK-3

）， phosphorylation of GSK-3

at Ser9 （p-Ser9-GSK-3

）， phosphorylation of GSK-3

at Tyr216 （p-Tyr216-GSK-3

）， total

-catenin （t-

-catenin）， phosphorylation of

-catenin （p-

-catenin） and CyclinD

proteins in hippocampus of mice in each group.

Result

Compared with the normal control group， mice in AD model group showed an obvious dementia state， which was characterized by significant declines in learning and memory ability， the number of

-catenin immunoreactive neurons in hippocampal CA1 area， the mRNA and protein expressions of t-

-catenin and CyclinD

， the protein expressions of p-Ser9-GSK-3

， and the ratio of p-Ser9-GSK-3

t-GSK-3

and p-Tyr216-GSK-3

t-GSK-3

in hippocampal region （

0.01）， and significant increases in the number of GSK-3

immunoreactive neurons in hippocampal CA1 area， the mRNA and protein expressions of t-GSK-3

， the protein expressions of p-Tyr216-GSK-3

and p-

-catenin， the ratio of p-

-catenin

-catenin in hippocampal region （

0.01 respectively）. Compared with the AD model group， the dementia symptoms of mice in HW group were significantly alleviated， and the number of

-catenin immunoreactive neurons in hippocampal CA1 area， the mRNA and protein expressions of t-

-catenin and CyclinD

， the protein level of p-Ser9-GSK-3

， the ratio of p-Ser9-GSK-3

t-GSK-3

in hippocampal region were all significantly increased （

0.01 respectively）， whereas the number of GSK-3

immunoreactive neurons in hippocampal CA1 area， the mRNA and protein expressions of t-GSK-3

， the proteins expressions of p-Tyr216-GSK-3

and p-

-catenin， the ratio of p-

-catenin

-catenin in hippocampal region were all significantly decreased （

0.01 respectively）， but the ratio of p-Tyr216-GSK-3

t-GSK-3

has no significant statistical difference.

Conclusion

HW shows the role of AD treatment， which can down-regulate the expression of GSK-3

in the hippocampus of AD mice and reduce its protein activity， and up-regulate the expression of

-catenin as well as increase its protein activity， so as to enhance the expression of downstream CyclinD

and promote the transcription of the target genes. Its mechanism may be related to the activation of Wnt

-catenin signal pathway.

关键词

Keywords

references

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Related Institution

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Effect of Huangjingwan on Expressions of Wnt/β-catenin Signal Pathway-associated Proteins in Hippocampus of Mice with Alzheimer's Disease Induced by D-galactose and Okadaic Acid with Learning and Memory Disorders

DOI：10.13422/j.cnki.syfjx.20201876

摘要

Abstract

关键词

Keywords

references